Extra-capillary fluid cycling system and method for a cell culture device

ABSTRACT

An extra-capillary fluid cycling unit for maintaining and cycling fluid volumes in a cell culture chamber includes a housing and a first flexible reservoir extra-capillary fluid reservoir disposed in the housing. The extra-capillary fluid reservoir is in fluid communication with a cell culture chamber. A second flexible reservoir is also located in the housing, the second flexible reservoir being in fluid communication with a pressure source. A sensor plate is movably disposed in the housing between the extra-capillary reservoir and the second reservoir, wherein the second reservoir is pressurized to move the sensor plate in relation to the extra-capillary reservoir to cause fluid cycling and maintain fluid volumes in the cell growth chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of:

International Application No. PCT/US2007/012053, filed May 21, 2007,which claims the benefit under 35 USC §119 of U.S. Application No.60/802,376, filed May 22, 2006, both of which are incorporated herein byreference in their entirety;

International Application No. PCT/US2007/012051, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety;

International Application No. PCT/US2007/012052, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety;

International Application No. PCT/US2007/012054, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006, both of which are incorporated herein by reference in theirentirety; and

International Application No. PCT/US2007/012042, filed May 21, 2007,which claims the benefit of U.S. Application No. 60/802,376, filed May22, 2006; both of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an extra-capillary (EC) fluid cyclingsystem for a cell culture device, and more particularly to an EC cyclingunit utilizing a non-rigid, EC reservoir fluidly connected to the cellculture device.

2. Description of the Related Art

The anticipated growth of personalized medicine will require newparadigms for the manufacture of therapies tailored to the needs ofindividual patients. The greatest challenge is expected to come in thearea of cell based therapies, especially when such therapies areautologous in nature. In such cases each cell or cell based product willneed to be manufactured from scratch for each patient. Manual methodsfor mammalian cell culture, by their nature, are prone to technicianerror or inconsistency leading to differences between supposed identicalcultures. This becomes especially evident as more and more autologouscells are expanded for personalized therapies. Patient-specific cells,or proteins, are subject to variation, especially when scaled beyondlevels that can be managed efficiently with manual methods.

In addition to being labor intensive, the stringent requirements forsegregation of each patient's materials from that of every other patientwill mean that manufacturing facilities will be large and complex,containing a multitude of isolation suites each with its own equipment(incubators, tissue culture hoods, centrifuges) that can be used foronly one patient at a time. Because each patient's therapy is a new andunique product, patient specific manufacturing will also be laborintensive, requiring not just direct manufacturing personnel but alsodisproportionately increased manpower for quality assurance and qualitycontrol functions.

Moreover, conventional approaches and tools for manufacturing cells orcell based products typically involve numerous manual manipulations thatare subject to variations even when conducted by skilled technicians.When used at the scale needed to manufacture hundreds or thousands ofpatient specific cell based therapies, the variability, error orcontamination rate may become unacceptable for commercial processes.

Small quantities of secreted product are produced in a number ofdifferent ways. T-flasks, roller bottles, stirred bottles or cell bagsare manual methods using incubators or warm-rooms to provideenvironments for cell growth and production. This method is very laborintensive, subject to mistakes and difficult for large scale production.Ascites production uses a host animal (usually a mouse) where theperitoneum is injected with the cells that express the product and areparasitically grown and maintained. The animals are sacrificed and theperitoneal fluid with the product is collected. This method is laborintense, difficult for large scale production and objectionable becauseof the use of animals. Another method is to inoculate and grow the cellsin a small stirred tank or fermenter. The tank provides theenvironmental and metabolic needs and the cell secretions are allowed toaccumulate. This method is costly in terms of facility support in orderto do a large number of unique cells and produces product at lowconcentration.

Another method is to use a bioreactor (hollow fiber, ceramic matrix,fluidizer bed, etc) as the cell culture device in lieu of the stirredtank. This can bring facilities costs down and increases productconcentration. Biovest International of Coon Rapids, Minn., has or hadinstruments using these technologies—hollow fiber, ceramic matrix,fluidized bed and stirred tanks.

Cell culturing devices or cultureware for culturing cells in vitro areknown. As disclosed in U.S. Pat. No. 4,804,628, the entirety of which ishereby incorporated by reference, a hollow fiber culture device includesa plurality of hollow fiber membranes. Medium containing oxygen,nutrients, and other chemical stimuli is transported through the lumenof the hollow fiber membranes or capillaries and diffuses through thewalls thereof into an extracapillary (EC) space between the membranesand the shell of the cartridge containing the hollow fibers. The cellsthat are to be maintained collect in the extracapillary space. Metabolicwastes are removed from the cultureware. The cells or cell products canbe harvested from the device.

Known EC reservoirs have typically been rigid. They are a pressurevessel and therefore require a sealed compartment with tubing portsadding to costs. A gas, typically air, is introduced through a sterilebarrier, generally a membrane filter, to control pressure in the vessel.Fluid level control has been limited to ultrasonic, conductive oroptical trip points, or by a load cell measuring the weight of thefluid. Reservoirs are expensive and difficult to manufacture. There islimited EC fluid level measurement accuracy-ultrasonic, conductive oroptical monitoring of fluid levels are commonly fouled by cell debris inthe reservoir. Alternatively, load cells are not a rugged design forreliable fluid level sensing.

These methodologies rely on costly, labor intensive off-line samplingand analysis or additional equipment to interface with the instrument orrequire the addition of a lactate probe and electronics to the culture.

Preparing the system to start the cell culture is also very laborintensive. The cultureware must be assembled and sterilized or probesmust be prepared, sterilized and aseptically inserted into thepre-sterilized portion of the cultureware. The cultureware assembly isthen loaded onto the instrument. A series of manual operations areneeded to check the integrity of the assembly, introduce fluid into thecultureware flow path, flush the toxic residuals from the cultureware,start the cultureware in a pre-inoculation mode, introduce factors intothe flow path getting it ready for the cells, inoculating the cells intothe bioreactor and starting the run (growth of the cell mass andeventual harvest of product).

Each unique cell or cell line must be cultured, cell products harvestedand purified separately. In order to do a large number of unique cellsor cell lines, a considerable number of instruments would be needed. Ifapplication of the cells or products for therapeutic purposes iscontemplated strict segregation of each cell production process would berequired. Consequently compactness of the design and the amount ofancillary support resources needed will become an important facilitiesissue. Moreover the systems currently available are general purpose innature and require considerable time from trained operators to setup,load, flush, inoculate, run, harvest and unload. Each step usuallyrequires manual documentation.

Accordingly, there is a need for an EC cycling device that is lessexpensive then the traditional rigid reservoirs and that providesaccurate EC fluid level measurement.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide an EC fluid cyclingunit that enables fluid level control without the use of expensiveultrasonics or load cells.

Another aspect of the present invention is to provide an EC cycling unithaving increased EC fluid level measurement accuracy and decreased celldebris in the reservoir, as well as easier assembly.

Yet another aspect of the present invention is an EC cycling unit havinga flexible reservoir.

Still another aspect of the present invention is to provide an ECcycling unit that costs less than rigid reservoirs. Also, a unit thathas a sealed EC circuit design, without a vented reservoir, inhibitscell contamination.

According to these and other aspects of the present invention, there isprovided an extra-capillary fluid cycling unit for maintaining andcycling fluid volumes in a cell culture chamber including a housing anda first flexible reservoir extra-capillary fluid reservoir disposed inthe housing, the extra-capillary fluid reservoir being in fluidcommunication with a cell culture chamber. A second flexible reservoiris also located in the housing, the second flexible reservoir being influid communication with a pressure source. A sensor plate is movablydisposed in the housing between the extra-capillary reservoir and thesecond reservoir, wherein the second reservoir is pressurized to movethe sensor plate in relation to the extra-capillary reservoir to causefluid cycling and maintain fluid volumes in the cell growth chamber.

According to these and other aspects of the present invention, there isalso provided a method for extra-capillary fluid cycling in a cellculture chamber comprising the steps of providing an extra-capillaryfluid cycling unit. The cycling unit including a housing, a firstflexible extra-capillary fluid reservoir disposed in the housing, asecond flexible reservoir located in the housing, and a sensor platemovably disposed in the housing between the extra-capillary reservoirand the second reservoir. A pressure source in communication with thesecond flexible reservoir is provided. A sensor in communication withthe pressure source is also provided, wherein the sensor plate includesan indicator in communication with the sensor. The pressure source isactivated to expand or contract the second reservoir. The sensor plateis moved to expand or contract the extra-capillary reservoir and theextra-capillary fluid is cycled through the cell culture chamber.

These and other features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of the preferred embodiment relative to the accompanieddrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the system for producing cells and/orcell derived products according to the present invention.

FIG. 2 is a cross-sectional view of a flexible hollow fiber bioreactorcell culture device according to the present invention.

FIG. 3 is a perspective view of the disposable culture medium module ofthe present invention.

FIG. 4 is a perspective view of the instrumentation base device of thepresent invention.

FIG. 5 is an interior view of the module of FIG. 3.

FIG. 6 is a perspective interior view of the back of the module of FIG.5.

FIG. 7 is a perspective view of the extra-capillary cycling unit of thepresent invention.

FIG. 8 is a flow diagram of the cycling unit of FIG. 7.

FIG. 9 is a perspective view of the fluid cycling control of the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1, the present invention provides a fully integratedsystem 10 for producing cells and cell derived products in a closed,self-sufficient environment. More specifically, the system allows forcell expansion and harvest of cells and their products with minimal needfor technician interaction. As will be described further herein, thedevice incorporates cell culture technology, for example, hollow fiberbioreactor perfusion technology, with all tubing components encased in asingle-use, disposable incubator. Following bioreactor inoculation withcells, the system follows pre-programmed processes to deliver media,maintain pH, maintain lactate levels, control temperature and harvestcells or cell-secreted protein. Standard or unique cell culture methodscan be programmed prior to bioreactor inoculation, such that, variouscell types or proteins can be expanded and harvested in an efficient,reproducible manner that is free of human error.

The system is based on cell growth chamber technology. Referring to FIG.2, a cell culture device or bioreactor 20 has a plurality ofsemi-permeable hollow fibers potted in a housing to create a spaceinside the fiber referred to as the intracapillary or IC space 22separate from a space outside the fibers referred to as anextracapillary or EC space 24. Fluid distribution between the IC space22 and EC space 24 occurs through the fiber pores, which can range insize from 10 Kd to 0.2 μm. Cells are placed on one side of the fiber,usually in the EC space, in a complete cell culture medium, which isusually the same medium used to expand cells prior to bioreactorinoculation (serum containing, serum-free, or protein-free medium).Cells are usually placed in the EC space when secreted protein is thedesired product. In some instances, when cells are the desired product,it may be beneficial to place cells in the IC space.

Medium is perfused through bioreactor 20 by circulating it through theIC space at a fast rate, in at 21 and out at 23. The medium is a liquidcontaining a well defined mixture of salts, amino acids and vitaminscontaining one or more protein growth factors. This serves to delivernutrients to the cell space and conversely, removes or prevents a toxicbuild-up of metabolic waste. Referring to FIGS. 10 and 11, during thiscirculation, medium is passed through an oxygenator 26 which serves toprovide pH control and oxygen for the cells and conversely, removecarbon dioxide from the culture. When the bioreactor 20 contains asmaller number of cells, just after inoculation, the oxygenator or gasexchange cartridge 26 is used to provide CO₂ and subsequently control pHof the culture environment. As cell number increases, the oxygenator isused to remove CO₂ which serves to enhance acid neutralization andcontrol the pH of the culture. It should be appreciated that otherculture vessels are contemplated by the present invention.

The system 10 provides significant efficiencies and cost reductionthrough its disposable component and enclosed operation. As such, celllines are contained in a closed system and continuously cultured withoutthe need for specialized, segregated clean rooms. This fully integratedapparatus eliminates the need for cleaning and sterilizationvalidations, as well as the need for hard plumbing associated withconventional cell culture facilities.

Referring again to FIG. 1, the system consists of two individual parts:an instrumentation base device 14 that is reusable and enclosedcultureware 12 that is used for a single production run and isdisposable. The instrument provides the hardware to support cell culturegrowth and production in a compact package. An easy-load multiplechannel peristaltic pump 16 moves fresh basal media into thecultureware, removes spent media, adds high molecular weight factor andremoves product harvest. An integrated cool storage area maintains thefactor and harvest at a low temperature (approximately 4° C.). Referringto FIG. 3, gas exchanger 26, in conjunction with a cultureware pH sensor30 controls the pH of the cell culture medium. Automated tube valvingdrives are used to control the cultureware flow path configuration toaccomplish the fluidic switching functions needed to initiate and do asuccessful run. Valves and sensors in the instrument control the fluidcycling in the cultureware module 12. A drive for fluid circulation isprovided.

The one-time use cultureware module 12 is provided pre-sterilized. It isdesigned for quick loading onto the instrument. The loading of thecultureware body makes connections to the instrument. A pump cassette32, which is physically attached to the tubing, allows the user toquickly load the pump segments. The design and layout minimizes loadingerrors. The cultureware enclosure 12 also provides an area that isheated to maintain cell fluid temperature.

Indicated in FIG. 3 and as shown in FIGS. 5-8, a fluid cycling unit 40maintains fluid volumes and cycling in the bioreactor and is included inthe cultureware. Sensors for fluid circulation rate, pH and a thermalwell for the instrument's temperature sensor are provided. The blendedgas from the instrument is routed to gas exchange cartridge 26 thatprovides oxygen and adds or removes carbon dioxide to the circulatedfluid to support cell metabolism. A magnetically coupled pump 60 (FIG.9) circulates fluid thru the bioreactor 20 and gas exchange cartridge26. The bioreactor 20 provides the cell space and media componentexchange and is also in the cultureware. Disposable containers forharvest collection are provided. Prior to the beginning of the culturethe operator attaches a media source, factor bag and spent mediacontainer to the cultureware before running. At the conclusion of therun the harvest containers are removed or drained, the media and spentmedia containers are disconnected, the pump cassette is unloaded, thecultureware body is unloaded and the used cultureware is placed in abiohazard container for disposal.

The system of the present invention has application in a regulated cellculture environment. It is anticipated that autologous whole celltherapies or patient-specific proteins (vaccines) therapies, would bytheir nature, require the simultaneous culture of numerous cell lines ina single facility. In addition to the segregation created through thisclosed culture approach, the apparatus is designed to support a standardinformation management system (MES) protocol. This capabilitycontributes to the creation of thorough batch records and verificationof culture conditions to ensure standardization, tracking and safety ofeach product. This capability facilitates the multi-product concept thatis pivotal to facilities involved with autologous or patient-specificproducts.

Referring to FIGS. 3 and 4, disposable cell culture module 12 isremovably attachable to device 14. The module requires multiplemechanical and electrical interfaces to the control instrumentation ofdevice 14. Module 12 has interface features integrated into the modulethat mate with instrument interface features in the device to allow fora single motion installation.

As shown in FIG. 4, the interface features of device 14 includecirculation pump 60 and a cycling sensor 34. Gas ports 36 communicatewith gas exchanger 26. One port 36 communicates with the input toexchanger 26 and the other port 36 communicates with the output of theexchanger. As viewed from the front, left port 36 is the exchanger outand right port 36 is the exchanger input. Gas ports 38 control pressureto cycling unit 40. One port 38 communicates with the IC chamber and theother port 54 communicates with an EC pressure bag that will describedfurther herein. The top port 54 is the IC reservoir pressurization portand the lower port 54 is the EC reservoir pressurization port.

During installation module 12 is aligned with the connections of thedevice 14 and the module is placed into the operating position as shownin FIG. 1. All mating interface features are functional. Referring toFIG. 6, when installed, certain features of the module 12 interface withdevice 14. Gas connectors 66 and 68 engage device gas ports 36 and 38,respectively, to allow gas to enter and exit module 12. Cycling unit 40communicates with cycling sensor 34 when the module is installed. Theabove mating connections facilitate the one-motion installation of themodule 12 on the device.

Referring to FIGS. 4, 7, 8 and 9, valves and sensor 34 in the instrumentbase control the fluid cycling in the cultureware module 12. Two opticalsensors 34A, 34B detect the low or high position of the cycling positionsensor indicator 52 (FIG. 7). This information is used by a predictivealgorithm to control the pressures applied to the IC chamber and ECpressure bag to effect cycling.

As shown in FIGS. 5-7, disposable cultureware module 12 includes fluidcycling unit 40 to maintain fluid volumes and cycling in the cell growthchamber or bioreactor 20. The present invention utilizes extra-capillary(EC) cycling in bioreactor 20 utilizing a non-rigid, first flexible ECreservoir 42 and a mechanical, second flexible reservoir 44 to causeelevated EC pressure. Reservoirs 42, 44 are movably located withinhousing 50, i.e., not physically attached to the housing, and areseparated by a movable sensor plate 46. Although free to expand orretract within housing 50, reservoirs 42, 44 are restricted in themaximum amount of expansion by the rigid side walls of the housing 50surrounding the same. The housing can be made from machined plastic orother comparable material. Sensor plate 46 can be made from aluminum,plastic or other comparable material. To facilitate even expansion orretraction of the respective reservoirs the side walls of housing 50 andsensor plate 46 are smooth or even.

As shown in FIG. 8, EC cycling is achieved by utilizing the EC,non-rigid reservoir to retain the excess fluid volume associated with anEC circuit. Flexible EC reservoir 42 is fluidly connected to bioreactor20 by connection 72 (FIG. 6) and EC circuit 76. Second flexiblereservoir 44 is fluidly connected at 74 (FIG. 6) to a pressure source70, that when expanded applies force against flexible reservoir 42 viaplate 46 to provide an elevated EC pressure to cause an ultra-filtrativecondition and force fluid into an intra-capillary (IC) circuit 48.Pressure source 70 can be supplied via an internal air pump containedwithin base device 14 and controlled via the fluid cycling control ofFIG. 9.

As will be described further herein, the bioreactor fibers arepermeable. A pressure differential from the EC side to the IC side ofthe cycling unit cause fluid to transmembrane into the opposite side andvice versa. Both reservoirs 42 and 44 can be made of a sealed flexiblematerial, for example, a plastic film bag made from a PVC/EVA(polyvinyl/acetate ethylene vinyl acetate) co-extrusion. The circulatingmedium (IC) is typically a standard growth medium that consists ofnutrients, vitamins, lipids, proteins, or carbohydrates required forcell proliferation or protein secretion. This medium may be substitutedor altered during the course of a culture to selectively affectproliferation, protein secretion, cell phenotype, cell signaling, orfacilitate cell removal from the bioreactor. The medium on the EC sideis the same as on the IC side, except that high molecular weightcomponents may be retained on the EC side because they can not permeatethe hollow fiber membrane. Cycling controls will force smaller molecularweight components of the medium from the EC side to IC side when the ECpressure is higher than the IC side.

Mechanical feed back position sensor indicator 52 is connected to sensorplate 46 and moves with the physical expansion and contraction of thefirst flexible reservoir 42. As shown in FIG. 7, sensor indicator 52 isattached to plate 46 at one end 53. Sensor indicator 52 includes a slot51 located therein. As plate 46 moves sensor indicator moves and isguided by element 64 located within slot 51. Element 64 is attached tothe housing. The position of indicator 52 is sensed by the positionsensor 34 and is used to control the force that is applied by secondflexible reservoir 44. As shown in FIG. 9, sensor 34 includes twooptical sensors 34A, 34B that detect the low or high position ofindicator 52. This information is used by a predictive algorithm tocontrol the pressures applied to the IC chamber and EC pressure bag toeffect cycling. Thus, if sensor 34 determines that indicator 52 is in alow position pressure applied to reservoir 42 may be reduced to enableEC reservoir to fill with fluid and vice-versa. It should be appreciatedthat an alternate mechanical force apparatus may be used instead of asecond flexible reservoir to cause pressure changes.

As shown in FIG. 8, during operation the pressure is increased in the ICcircuit 48 by pressurizing an IC reservoir 54. This pressure causes anultra-filtrative condition that forces fluid transmembrane across thesemi-permeable matrix of the bioreactor 20. The fluid is then forcedthrough the connect tubing, through a flow control valve 56 and into theEC reservoir 42. The expanding EC reservoir 42 forces sensor plate 46toward pressure reservoir 44 and compresses it. Sensor plate 46activates external position sensor 52 when EC reservoir 42 has filledenough to expand to the EC upper level. External position sensor 34senses this position and the pressure in the IC reservoir 54, isdecreased and the pressure in the pressure reservoir 44 is increased.This causes an ultra-filtrative condition and forces fluid out of the ECreservoir through a control valve 58, transmembrane across the matrix ofbioreactor 20 and into the IC circuit 48. The sensor plate moves theexternal position indicator 52 and the sensor 34 senses when ECreservoir 42 has contracted to the EC low level.

The EC cycling unit of the present invention offers fluid dynamics tocause fluid flow in the EC space, thus minimizing nutrient and metabolicwaste gradients that may be detrimental to the cells. It also providesfluid level control without the use of ultrasonics or load cells and isnot affected by cell debris. The flexible reservoirs of the cycling unitof the present invention are considerably less expensive and are suitedfor disposable applications. The sealed EC reservoir with cycling alsolimits contamination and isolates the cells.

In the bioreactor perfusion loop of FIG. 8, the growth media is pumpedfrom IC reservoir 54 via pump 16, 60, circulated to gas exchangecartridge 26, pH sensor 30, hollow fiber bioreactor 20, and then back toreservoir 54. Blended gases are passed through the membrane gas exchangecartridge that oxygenates the media and removes unwanted CO₂. PerHenry's Law, the CO₂ levels in the gas phase or air side of the gasexchange cartridge 26 is in equilibrium with the liquid phase of themedia. The discharge end of the gas exchange cartridge is monitored witha CO₂ sensor.

At present, the system of the present invention fully integrates theconcept of disposable cultureware into automated process control formaintaining and expanding specialized (autologous or other) cell linesfor a duration for 30 days or more. To accomplish this, the system ofthe present invention was designed for EC space fluid flow that enhancescell growth in high density perfusion culture, yet remains completelyclosed and disposable. The integrated pre-assembled cultureware, whichconsists of all tubing, bioreactor, oxygenator, pH probe, is enclosed ina single unit that easily snaps into the apparatus. In addition to thiserror-proof, quick-load design, the entire cultureware unit enclosed bythe casing becomes the cell culture incubator with temperature controlregulated through automated process control of the instrument. Pumps andfluid control valves facilitate disposability and error-proofinstallation, eliminating the possibility of technician mistakes.Finally, during the course of any culture, the closed system hasrestricted access except for trained and authorized personnel.Manipulations or sampling, outside of program parameters, requirepassword and bar code access before they can be implemented.

Each unique cell line must be cultured, cell secretions harvested andpurified separately. In order to manage a large number of unique celllines, as for example might be required for the production of largenumbers of autologous cell therapeutic products or large numbers ofunique monoclonal antibodies, a considerable number of instruments wouldbe needed. Compactness of the design and the amount of ancillary supportresources needed become an important facilities issue. Small stirredtank systems require a means of steam generation and distribution (forsteam-in-place sterilization) or autoclaves to sterilize the vessels andsupporting plumbing. To support a large number of units becomes alogistics problem for the facility. The system of the present inventionhas no such requirement. Larger scale cell culture is historically donein segregated steps that often require separate types of equipment.Manual handling, storage and tracking is needed for all these steps asthe culture expands and product is harvested. The method of the presentinvention integrates these steps into a continuous, fully integratedsequential process. This eliminates the handling risk and facilitatesthe data gathering required for thorough documentation of the entireprocess.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. It ispreferred therefore, that the present invention be limited not by thespecific disclosure herein, but only by the appended claims.

1. A fluid cycling unit for maintaining and cycling fluid volumes in achamber comprising: a housing; a first flexible fluid reservoir disposedin the housing, said first fluid reservoir being in fluid communicationwith a chamber; a second flexible reservoir located in the housing, saidsecond flexible reservoir being in fluid communication with a pressuresource; and a sensor plate movably disposed in the housing between firstand second reservoirs, wherein said second reservoir is pressurized tomove the sensor plate in relation to the first reservoir to cause fluidcycling and maintain fluid volumes in the chamber.
 2. The fluid cyclingunit of claim 1, further comprising a sensor indicator connected to thesensor plate, wherein physical expansion and contraction of the first,flexible reservoir moves the sensor indicator within the housing.
 3. Thefluid cycling unit of claim 2, further comprising a sensor incommunication with the pressure source, wherein the sensor indicatordrives the sensor to expand or reduce the pressure within the secondflexible reservoir.
 4. The fluid cycling unit of claim 1, wherein saidchamber comprises a hollow fiber bioreactor that provides cell space andmedia component exchange.
 5. An extra-capillary fluid cycling system formaintaining and cycling fluid volumes in a cell culture chamber of acell culture environment comprising: an extra-capillary fluid cyclingunit, the cycling unit including a housing, a first flexible reservoirextra-capillary fluid reservoir disposed in the housing, a secondflexible reservoir located in the housing, and a sensor plate movablydisposed in the housing between said extra-capillary reservoir and saidsecond reservoir, wherein the extra-capillary fluid reservoir is influid communication with the cell culture chamber; a pressure source incommunication with said second flexible reservoir; a sensor incommunication with the pressure source, wherein the second reservoir ispressurized to move the sensor plate in relation to the extra-capillaryreservoir to cause fluid cycling and maintain fluid volumes in the cellgrowth chamber.
 6. The extra-capillary fluid cycling system of claim 5,further comprising a sensor indicator connected to the sensor plate,wherein physical expansion and contraction of the extra-capillaryreservoir moves the sensor indicator within the housing to control theactivation of the pressure source.
 7. The extra-capillary fluid cyclingunit of claim 6, wherein said cell growth chamber comprises a hollowfiber bioreactor that provides cell space and media component exchange.8. A method for extra-capillary fluid cycling in a cell culture chambercomprising the steps of: providing an extra-capillary fluid cyclingunit, said cycling unit including a housing, a first flexibleextra-capillary fluid reservoir disposed in the housing, a secondflexible reservoir located in the housing, and a sensor plate movablydisposed in the housing between said extra-capillary reservoir and saidsecond reservoir; providing a pressure source in communication with saidsecond flexible reservoir; providing a sensor in communication with thepressure source, wherein said sensor plate includes an indicator incommunication with said sensor; activating said pressure source toexpand or contract said second reservoir; moving said sensor plate toexpand or contract said extra-capillary reservoir; and cycling theextra-capillary fluid through the cell culture chamber.
 9. The method ofclaim 8, wherein the step of cycling the extra-capillary fluid comprisesmoving the fluid through a cell space of the chamber and exchangingmedia component.
 10. The method of claim 9, further comprising the stepof activating the sensor with said indicator, wherein physical expansionand contraction of the extra-capillary reservoir moves the sensorindicator within the housing to control the activation of the pressuresource.